RECOMBINANT ANTIGENS FROM MUMPS VIRUS AND THEIR USE IN VACCINES

Abstract: The invention provides recombinant DNA encoding an antigen of mumps virus selected from the group comprising: (a) the full length fusion protein (F), complete with signal peptide(s) and membrane anchor domain (a) (designated herein as Fs+a+); the full length attachment protein or hemagglutinin-neuraminidase (HN); the full length nucleocapsid protein (NP); (b) the truncated fusion protein lacking the membrane anchor domain (herein designated Fs+a-); (c) a hybrid protein derived by fusion of the F, HN and/or NP proteins or portions thereof. Also within the scope of the invention are vectors comprising the DNA of the invention and a host such as a vaccinia virus, mammalian cell or bacterial cell transformed or transfected with the vector; and proteins expressed by the hosts.

Description: RECOMBINANT ANTIGENS FROM MUMPS VIRUS AND THEIR USE IN VACCINES The Mumps virus belongs to the paramyxoviridae, subclass paramyxovirus. It is a pathogen causing the contagious infantile illness which consists of the inflammation of parotid glands. During the incubation period following infection, the virus replicates in the respiratory epithelium then disseminates into secretory ducts of the parotid glands. Other glands may become infected thereafter and numerous cases of meningitis have been reported. Among complications related to the infection, encephalitis is a serious one, with a mortality rate of about 1%; deafness cases have also been reported. A vaccine against Mumps is available: it is made of an attenuated live virus, produced by culturing infected embryonic chicken cells. The vaccine leads to the seroconversion in vaccinated individuals and protects against infection in more than 95% of seronegative persons. The vaccine thus reduced significantly the frequencies of complications. In a number of cases, however, viral infection is not detected because the effects remain subclinical. Young children and aged people are most likely to develop complications from Mumps infection. In view of the inherent risks related to the use of attenuated live vaccines, such as die potentiation of the illness upon natural surinfection in vaccinated individuals, it is desirable to improve the safety of the vaccine, particularly for the groups at risk. The present invention addresses this issue by providing systems to produce recombinant Mumps proteins in host cells, especially eukaryotic cells, particularly the F, HN and NP proteins and fusions proteins derived therefrom. The invention also relates to methods of constructing the said proteins, intermediates for use therein and recombinant proteins which may be obtained from the intermediates. In a particular embodiment of the invention, the Mumps virus NP protein has also been expressed in bacteria. Recombinant proteins of the invention have potential utility in the development of subunit vaccines for the prevention of Mumps virus infections. The fusion protein F of Mumps virus contains 538 amino acid residues; amino acids 1 to 26 correspond to the signal peptide and residues 483 to 512 to the membrane anchor domain. The molecule presents 7 potential sites for glycosylation. The F protein is synthesized as a 65-74 KDa precursor (F 0 ) which undergoes proteolytic maturation to yield the F j (58-61 FDa) and F2 (10-16 KDa) subunits linked via disulfϊde bridges. The protein F is involved in cell fusion during viral infection, carries an haemolysin activity and plays a role for viral penetration into cells. It does not however carry the antibody dependent cellular cytotoxicity (ADCC) a observed for another Mumps virus glycoprotein, HN (see below). The protein HN (molecular weight 74-80 KDa), carries hemagglutinin and neuraminidase activities which are involved in virus attachment to cells and in the disruption of the host cell membranes. Protein HN ('attachment protein' or hemagglutinin-neuraminidase) generates neutralizing antibodies and appears important for the development of ADCC. Protein HN is composed of 582 amino acids; it carries a N-terminal anchor domain (residues 33 to 52) and 9 potential sites for glycosylation. The nucleocapsid protein NP is associated to the viral RNA and is involved in its transcription process. By analogy with influenza viruses, the NP protein of Mumps virus might be important for the development of cellular immunity. The NP protein is made of 553 amino acids; it has a molecular weight of 72 KDa and is phosphorylated. According to the present invention there is provided recombinant DNA encoding an antigen of mumps virus selected from the group comprising: (a) the full length fusion protein (F), complete with signal peptide (s) and membrane anchor domain (a) (designated herein as Fs+a+); the full length attachment protein or hemagglutinin-neuraminidase (HN); the full length nucleocapsid protein (NP); (b) the truncated fusion protein lacking the membrane anchor domain (herein designated Fs+a-); and (c) a hybrid protein derived by fusion of the F, HN and/or NP proteins or portions thereof. In particular embodiments of the invention DNA encoding the hybrid protein encodes: (i) the attachment protein lacking the 5' membrane anchor domain fused to the signal domain of the fusion protein (herein designated s+FHNa-); or (ii) the hybrid protein herein designated s+FHNa-xFa-; or (iii) the fusion protein lacking the 3' membrane anchor domain fused to the attachment protein lacking the 5' membrane domain (herein designated Fs+a-xHNa-). Preferably the recombinant DNA according to the invention is a vector, advantageously an expression vector, which is suitable for tranfecting or transforming a suitable host so that quantities of the protein encoded by the DNA of the invention may be obtained. In one aspect the recombinant vector according to the invention is a vaccinia transfer vector, for example a vector derived from pULB5212 as described hereinbelow. Particular vectors of this type are designated herein pNIV3205, pNIV3208, pNIV3213 and pNIV3232. In another aspect the recombinant vector according to the invention is one which is suitable for transfecting mammalian cells, especially Chinese Hamster Ovary (CHO) cells. Advantageously a vector of this type may be derived from a glutamine synthetase vector, for example a vector known as pEE14 as described hereinbelow. A particular vector according to the invention is pEE14s+FHNa-. In yet another aspect the recombinant vector according to the invention is one which is suitable for transforming bacterial cells such as E.coli. Suitable vectors of this sort may be derived, for example, from the well known vector pUC19. In another aspect of the invention there is provided a host transformed or transfected with a vector according to the invention. The host may suitably be selected from a vaccinia virus; a mammalian cell or a bacterial cell. The invention also provides a method for preparing a recombinant protein encoded by DNA according to the invention comprising: (a) expressing the said DNA in a suitable recombinant host as described above; and (b) isolating the protein produced by standard techniques, for example as described hereinbelow. Certain proteins produced by the method of the invention are novel and accordingly these form a further aspect of the invention. Particular proteins include: a truncated fusion protein lacking the membrane anchor domain (herein designated Fs+a-); a hybrid protein derived by fusion of the F, HN and/or NP proteins or portions thereof, especially: (i) the attachment protein lacking the 5' membrane anchor domain fused to the signal domain of the fusion protein (herein designated s+FHNa-); (ii) the hybrid protein herein designated s+FHNa-xFa-; (iii) the fusion protein lacking the 3' membrane anchor domain fused to the attachment protein lacking the 5' membrane domain (herein designated Fs+a-xHNa-). Proteins which are obtainable by the method of the invention have potential as vaccines when admixed with a suitable carrier. Accordingly in a further aspect the invention provides a vaccine composition against mumps infection comprising an immunoprotective amount of a protein according to the invention or prepared according to the process of the invention together with a pharmaceutically acceptable carrier. The protein in such a vaccine composition is adantageously the F or HN protein. The term 'immunoprotective' refers to the amount necessary to elicit an immune response against a mumps challenge such that disease is averted or mitigated and transmission of the disease is blocked or delayed. Vaccine preparation is generally described in New Trends and Developments in vaccines, Voller et al (eds), University park Press, Baltimore, Maryland, 1978. The amount of protein of the present invention present in each vaccine dose is selected as an amount which induces an immunoprotective response without significant adverse side effects in typical vaccines. Such amount will vary depending on which specific immunogen is employed and whether or not the vaccine is adjuvanted. Generally it is expected that each dose will comprise l-lOOOug of protein, preferably 1 - 200ug. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects will preferably receive a boost in about 4 weeks, followed by repeated boosts every six months for as long as a risk of infection exists. A further aspect of the invention provides a method of preventing mumps infection in humans which method comprises administering to a subject in need thereof an immunologically effective amount of the protein of the invention or the protein prepared by the method of the invention. The invention also provides the use of a protein according to the invention or a protein prepared by the method according to the invention for the preparation of a vaccine composition for the prevention of mumps infection in human subjects. The following examples and the attached Figures (explained below) illustrate the invention. Examples Examples 1 Vector Construction A) For transfer into vaccinia virus 1) The Fusion protein Fs+a+ Starting from plasmid pMFl, a 900 bp cDNA clone encoding the C-terminal half of the F protein (received from Dr. E. Norrby, Karolinska Institute, Stockholm, Sweden, and described in Elangofit al, 1989, J.Gen. Virology 70, 8001-807), we reconstructed the cDNA coding for the complete Fs + a + protein, i.e. containing both signal peptide and membrane anchor domains. To this end, the viral genomic RNA, extracted from Mumps viruses, was copied into complementary single strand DNA using as primer as oligodeoxynucleotide (po82) corresponding to bases 1 to 20 of the Mumps FmRNA and having the following sequence (5'-AAG CCT AGA AGG ATA TCC TA-3'). Then, the single stranded cDNA was amplified by the polymerase chain reaction using as primers po82 and pstFMl or pstFM2 and pF1013. The first set of primers generates after amplification a double stranded cDNA corresponding to bases 1 to 769 of the Mumps FmRNA and the second set of primers a cDNA fragment covering bases 748 to 1035. The sequence of the pstFMl, pstFM2 and pD1013 primers is as follows: pstFMl 5' CCA CTG CAG GTG TCA TAC TTC C 3' PstI 769 748 pstFM2 5' GGA AGT ATG ACA CCTGCA GTC G 3' PstI 748 769 pF1013 5' ACA ATT CTT AGC TGG ATA GCG CC 3' 1035 1013 The amplified cDNA fragments were then separately cloned into the Hindll- Esil sites of plasmid pUC19 and sequenced using the dideoxynucleotide method (Sanger ej al, 1977, P.N.A.S.74, 5463-5467). Both DNA fragments were ligated together through their common P&I site and cloned into the EcoRI site of plasmid pUC19; this leads to the intermediate construct pNIV3204. Plasmid pNIV3204 was cleaved by the EcoRI and Haell enzymes to recover the 5' end of the F cDNA. The 3' end of the F cDNA was recovered by digesting plamid pMFl witgh PstI and Haell. The complete F cDNA is reconstituted by ligating the 5' and 3' parts via their common Haell restriction site and introduced into the EcoRI-PstI sites of the cloning vehicle pUC19, yielding the plasmid pNIV3214 (Fig. 1A). From there, the cDNA module (1701 bp) encoding the FS + a + protein was recovered by cleavage with enzymes S al and Ahaiπ and introduced into the S al site of the vaccinia transfer vector pULB5213, which is a derived of the standard vaccinia vector pSCl 1 (Chakrabati ej aL 1985, Molecular and Cellular Biology 5, 3403-3409). The resutling plasmid, pNIV3205, is illustrated in Fig. IB. 2) The Attachment protein HN Starting from plasmid pMHl, a 1000 bp cDNA clone encoding the C-terminal part of the protein HN (received from Dr. E. Norrby, Karolinska Institute, Stockholm, Sweden and published by Kovamees el aL 1989, Virus Research 12,87-96), we reconstructed a cDNA clone corresponding to the complete HN proetin of the Mumps virus. To this end, the procedure followed the one described above for the F protein. In short, Mumps viral RNA was first copied into single stranded cDNA using the po83 oligodeoxynucleotide as primer (5' AAG CCA GAA CAG ACT TAG GAT 3'). This cDNA was then amplified by PCR with two sets of primers to generate a first double stranded cDNA fragment corresponding to bases 79 to 629 of the HNmRNA (primers pHN-13 and EcoHNMl) and a second one spanning bases 603 to 1005 (primers EcoHNM2 and pH982). The sequence of these primers is as follows: pHN-13 5' GAC CCG GG CCACC ATQ GAG CCC 3' Smal Kozak Metl sequence, initiation consensus EcoHNMl 5" GAT GGA ATT CTT GTG CAA CCA TTG 3' EeoRI 626 603 EcoHNM2 5' CAA TGG TTG CAC AAG AAT TCC ATC 3' EcoRI 603 626 pH982 5' CCC CAC TCC TGG CAC CAA AGT AGC 3' 1005 982 The amplified cDNA fragments were cloned into the Hindll-EcoRI sites of plasmid pUC19, sequenced by the Sanger method (see supra) and then ligated together via their EcoRI site into the pUC19 cloning vehicle. The resulting intermediate construct, pNIV3207, thus carries the 5' end of the HN coding sequence which can be excised from pNIV3207 by digestion with Hindi and Banl. The 3 * end of the HN cDNA was recovered by digesting plasmid pMHl with Banl and Hindlll. The complete HN cDNA is reconstituted by ligating the 5' and 3' parts via their common Banl site and introduced into the Hin i and Hindu sites of plasmid pUC19, yielding the plasmid pNTV3215 (Fig.2A). The cDNA module (1779bp) encoding the complete HN protein was recovered by cleavage with enzymes Hindlll and Smal and introduced, blunt-ended, into the Smal digested vaccinia transfer vector pULB5213 (see supra) . The resulting plasmid, pNIV3208, is illustrated in Fig.2B. 3) The nucleocapsid protein NP The cDNA coding for the complete Mumps NP protein was assembled from two partial cDNA clones, pMNl and pMN2 (obtained from Dr. E. Norrby, Karolinska Institute, Stockholm, Sweden and published by Elango, 1989, Virus Research 12.77-86). Plasmid pMNl carries an insert of 1700 bp which lacks the 5' end of the NP cDNA; pMN2 has a cDNA insert of 1700 bp which does not encompass the 3' end of the molecule. pMNl was digested by PstI to recover a 1100 bp fragment which was subcloned into the PstI site of the plasmid vector pUC19. This intermediate construction was digested by Asp718I and EcoRV to recover a fragment that encodes the 248 C-terminal amino acids of the NP protein and carries the stop codon. pMN2 was digested with HgiAI and PvuII to recover a 1361 bp fragment spanning amino acid residues 6 to 458. A synthetic double stranded nucleotide (Muml Mum2) was prepared in order to reconstruct the ATG initiation codon (Metl) and the sequence coding for amino acid 2 to 5 of the NP protein (Fig.3A). This synthetic fragment was flanked by a BamHI site (5') and a HgiAI site (3'); it also contained the KOZAK consensus (Kozak, 1984, Nature 308. 241-246) which is optimal for translation initiation. The synthetic adaptor and the 1361 bp DNA fragment recovered from pMN2 were assembled and cloned into the plasmid vector pUC19. This intermediate construction was digested by Asp718I and EcoRV. a 466 bp fragment was eliminated and replaced by the Asp718I and EcoRV fragment that encodes the 248 C-terminal amino acids derived from pMNl. This leads to plasmid pNTV3216 which contains a 1699 bp coding module for the complete NP protein carried by vector pUC19 (Fig.3B). Plasmid pNIV3216 was then digested with BamHI and Bell to exdse the coding cassette (1682 bp) which was inserted blunt-ended into the Smal site of the vaccinia transfer plasmid pULB5213 (see supra, yielding the final construct pNIV3213 (Fig.3C). 4) The fusion protein Fs + a÷ lacking the membrane anchor domain Plasmid pNIV3214 (see above), a cDNA clone encoding the full length F protein, was digested with Nsil and Sphl. A 250 bp Nsil-SphI fragment encoding die 80 C-terminal amino acids of the F protein, carrying the membrane anchorage domain (residues 483-512) and the stop codon, was elimated. A synthetic double stranded nucleotide (Fa " mul/Fa " mu2) was prepared in order to reconstruct the sequence coding for amino acid residues 459 to 462 and a stop codon (Fig.4A). This synthetic fragment was flanked by a Nsil site (5') and a Sphl site (3'). The synthetic adaptator was cloned into the Nsil and Sphl site of pNIV3214. The resulting plasmid, pNIV3220, encodes amino acid residues 1 to 462 followed by a stop codon, cloned into the pUC19 vector. In order to create a KOZAK consensus sequence (see supra), plasmid pNIV3220 was digested by BsphI and Asp718I: protruding ends were filled by Klenow polymerase then ligated. This creates plasmid pNIV3221 which contains the coding sequence for residues 1 to 462 of the F protein followed by a stop codon and with a KOZAK consensus around the initiation codon (Fig.4B). Plasmid pNIV3221 was digested with Asp718I and Sphl. a 1402 bp fragment was blunt-ended and inserted by ligation into the Smal site of pULB5213 (see supra). The resulting plasmid pNIV3226 is illustrated in Fig.4C. 5) The hemagglutinin-neuraminidase lacking the 5' membrane anchor domain fused to the signal domain of the fusion protein. s + FHNa- Plasmid pNIV3215 (see above), a cDNA clone encoding the entire HN protein, was digested with PvuII: a 1700 bp fragment encoding amino acid residues 79 to 582 and carrying a stop codon was purified. Plasmid pNIV3221 encoding amino acid residues 1 to 462 of the Fusion protein (F) (see supra) was digested with HindTII. A 2900 bp fragment, carrying the pUC19 sequence and the sequence coding for amino acid residues 1 to 51 of the Fusion protein, was blunt-ended and ligated to the 1700 bp PvuII fragment encoding amino acid residues 79 to 582 of the HN protein followed by a stop codon into the pUC19 vector (Fιg.5A). Plasmid pNIV3222 was digested by Asp.7181 and Hindlll; a 1694 bp blunt-ended DNA piece was purified and inserted by ligation into the Smal site of pULB5213 (see supra). The resulting plasmid pNIV3227 is illustrated in Fig.5B. 6) The hybrid protein s+FHNa-xFa- Plasmid pNTV3221 (see supra) which contains the sequence coding for the F protein lacking the anchor domain was digested with Hindlll. A 1246 bp blunt-ended fragment that encodes amino acid residues 52 to 462 of the F protein was purified. Plasmid pNIV3222 (see supra) which encodes amino residues 1 to 51 of die fusion protein, an irrelevant threonine (aa 52) and amino acid residues 79 to 582 of me HN protein followed by a stop codon, was digested by Nhel which cleaves on the 3' side of the codon specifying amino acid residues 576. Plasmid pNIV3222, digested by Nhel and blunt-ended, was then assembled by ligation to the 1246 bp fragment mat encodes amino acid residues 52 to 462 of the F protein. The resulting plasmid pNTV3223 thus encodes, in a pUC19 vector, amino acid residues 1 to 51 (encompassing the signal of the F protein), an irrelevant threonine, amino acid residues 79 to 576 of the HN protein, an irrelevant glutamine and amino add residues 53 to 462 of the F protein followed by a stop codon (Fig.6A). Plasmid pNIV3223 was digested by Asp718I and Hindlll: a 2945 bp DNA fragment was ligated blunt- ended into the Smal site of the pULB5213 (see supra). The resulting plasmid is illustrated in Fig.6B. 7) The fusion protein lacking the 3' membrane anchor domain fused to the attachment protein lacking the 5' membrane anchor domain Fs+a-xHNa- Plasmid pNIV3215 (see supra) which encodes the entire HN protein was digested by Hind III, protruding ends were filled by Klenow polymerase, and by Saς I; a 1762 bp fragment was ligated to the pUC 19 vector digested by Sma I and S £ I creating plasmid pNIV 3229. A synthetic double strand adaptor (fhnoligol/fhnoligo2) was prepared in order to construct a junction between the F and HN coding sequence allowing a continuous open reading frame (Fig. 7A). Plasmid pNIV 3229 was digested by Kpn I and Bbs I, a 229 bp fragment encoding amino acid residues 1 to 66 of the HN protein was eliminated and replaced by the synthetic adaptor, this leads to pNIV 3230. Plasmid pNIV 3221 (see supra) which encodes the F protein lacking the anchor domain was digested by Kpn I and P I. A 1387 bp fragment was purified and ligated to the pNIV 3230 digested by Kpn I and N≤i I. This creates plasmid pNIV 3231 which contains, carried by vector pUC 19, a module coding for amino acid residues 1 to 460 of the F protein, followed by an histidine and amino acid residues 63 to 578 of the HN protein followed by a stop codon (Fig. 7B). Plasmid pNIV 3231 was digested by Asp718I and Sphl to excise a 3007 bp coding module which was inserted blunt-ended into the Sma I site of the vaccinia transfer plasmid pULB 5213 (see supra) yielding pNIV 3232. B) For transfection into CHO cells 1) The fusion protein Fs + a ~ lacking the membrane anchor domain Plasmid pNIV3221 (see above) was digested with Asp718I and Hindlll: a 1402 bp blunt-ended fragment was purified and cloned into the Smal site of the glutamine synthetase (GS) vector, pEE14 (Cockett el ai. 1990, Bio/Technology & 662-667). The resulting plasmid pEE14-Fa " contains the fusion protein lacking the anchor domain under me control of the major immediate early promoter of me human cytomegalovirus (hCMV-MEE) (Fig.4D). 2) The hemagglutinin-neuraminidase lacking the 5' membrane anchor domain fused to the signal domain of the fusion protein. s + FHNa~ Plasmid pNIV3222 was digested by Asp718I and Hindlll: a 1694 bp blunt- ended DNA piece was purified and inserted by ligation into the Smal site of the pEE14 vector (see supra). The resulting plasmid, pEE14 s + F HNa", contains under the control of the hCMV promoter, the sequence for amino acid residues 1 to 51 of the F protein (encompassing the signal region of the F protein), for an irrelevant threonine and for amino acid residues 79 to 582 of the HN protein (Fig.5C). 3) The hybrid protein s+FHNa-xFa- Plasmid pNIV3223 (see above) was digested by Asp718I and HindHI; a 2945 bp DNA piece was recovered and ligated blunt-ended into die Smal site of the pEE14 vector (see supra). The resulting plasmid, pEE14 s + F HNa'xFa " , contains under die control of the hCMV promoter, the sequence coding for the signal region of the F protein (aa 1 to 51), an irrelevant glutamine, the F protein lacking its signal and its anchor domain (Fig.6C). 4) The fusion protein lacking the 3' membrane anchor domain fused to the attachment protein lacking the 5' membrane anchor domain. Fs+a-xHNa- Plasmid pNIV 3231 (see above) was digested by Asp718I and S JiI to excise a 3007 bp fragment which was ligated blunt ended into die £ma I site of the pEE14 vector (see above). The resulting plasmid, pEE14 Fs+a-xHNa-, contains under die cotrol of the hCMV promoter, die sequence coding for the F protein lacking its membrane anchor domain (aa 1 - 460) and an histidine fused to d e HN protein lacking its membrane anchor domain (aa 63 - 578) followed by a stop codon (Fig. 7C). C) For transformation of bacteria The NP protein Plasmid pNIV3216 (see above), which contains die cDNA for d e entire NP protein, in the pUC19 vector, was digested by BamHI. A 1682 bp blunt-ended fragment was purified and cloned into die blunt-ended BamHI site of the E.coli expression vector p AS 1 (Rosenberg si al, in Methods in Enzymology: Wu,R. £l aL Eds., Vol.101.pp. 123-138, Academic Press, New York, 1983). The resulting plasmid pNTV3217 contains, under die control of the bacteriophage lambda PL promoter, the sequence for 7 irrelevant amino acids (including an ATG initiation codon) fused to die sequence of die complete NP protein. (Fig.8). Example 2 Expression in eukaryotic cells A) via vaccinia virus recombinants Recombinant transfer plasmids, pNV3205, pNIV3208, pNIV3213 and pNTV 3232, were transfected into vacdnia-infected CV-1 cells and recombinant viruses were isolated after Bromo-uridine selection and plaque purification on die basis of dieir blue colour in the presence of X-gal. They will be referred to as VV3205, VV3208, VV3213 and VV3232 respectively. The RAT2 TK' strain was of the WR type (origin Borisewitz, U.K.). The procedure follows that one previously described for the obtention of vaccinia virus recombinants (Mackett, M. and Smith, G.L., J.Gen. Virology £7_, 2067- 2082, 1986; Mackett, M., Smith, G.L. and Moss, B., J. Virology 4£, 857-864, 1984). 1) Fusion protein Fs + a* The recombinant vaccinia virus, VV3205, was used to infect CV-1 cells in culture at a multiplicity of infection 1 (m.o.i=l). Infected cells (about 3.10 5 per assay) and spent culture medium (about 2ml) were collected between 16 and 48 hours post infection. The presence of the Fs + a + protein was demonstrated by immunoprecipitation on cell extracts and spent culture medium. Immunoprecipitation experiments were performed as follows. In short, the recombinant product was labelled in vivo by growing the recombinant vaccinia virus in the presence of 35 S-methionine. Labelled samples, cell extracts and medium were incubated successively widi a mixture of monoclonal antibodies (Mab 2.049, 2.159, 5.369, 5.414, 5418, 5.439, Orvell, J.Immunol., 1494122, 2622-2629, received from Dr. E.Norrby, Karolinska Institute, Stockholm, Sweden) and rabbit antimouse serum; specific complexes were then recovered by binding to sepharose-bound protein A. Immunoprecipitates were analysed onto 12% SDS-polyacrylamide gels, which were then autoradiographed after drying. The intracellular recombinant product migrated on SDS-PAGE as a 70 KDa (F c ) precursor and a 60 KDa (F,) subunit in reduced conditions. The product found in die spent culture medium was present exclusively under the form of the Fj subunit. The F 2 subunit cannot be detected in die assay due to die absence of methionine in its sequence. Alternatively, die immunoprecipitation experiments were also done widi 3 H-glucosamine. In this case, the cell extracts were incubated widi Mab 2.019 and 2.159 (see supra). Specific complexes were recovered by binding to Sepharose-bound protein A and immunoprecipitates were analyzed onto 17% SDS-polyacrylamide gels. The recombinant product migrated as a 60 kDa bands in reduced conditions representing the glycosylated and proteolytically cleaved Fj and F 2 subunits, respectively, of die fusion glycoprotein (Fig.9, lane 3). 2) Hemagglutinin-neuraminidase HNs+a+ The recombinant vaccinia virus, VV3208, was used to infect CV-1 cells in culture. The expressed product was analyzed by radioimmuno-predpitation. The procedures followed for labelling with 35 S-metiιionine were the same as those described above, except for the mixture of monoclonal antibodies (Mab 741, 743, 1.933, 2.072, 2.073, 2.075, same ref. as in example 2-A1, provided by Dr.E.Norrby, see supra) used for immunoprecipitation. The recombinant protein was found exclusively in die cell extracts at a 80 KDa product. For labelling the cell extracts with 3 H-glucosamine, the procedures were also the same as those described above, except that Mab 2.075 was used instead of a mixture of Mab. The recombinant was found as a glycosylated monomer of apparent size of 75kDa (Fig.9, lane 2). 3) Nucleocapsid protein The recombinant vaccinia virus, VV3213, was used to infect CV-1 cells. The expressed product was identified by Western blotting. The cell extract and spent culture medium were resolved onto 12% SDS-polyacrylamide gels. After transfer onto nitrocellulose membranes, separated proteins were probed widi a mixmre of monoclonal antibodies (Mab 705, 728, 2.099, ref. as in example 2,A,1, provided by Dr.E.Norrby, see supra). Complexes were detected using a rabbit antimouse IgG conjugated to alkaline phosphatase and the appropriate chromogenic substrate, according to standard procedures. The recombinant product was exclusively found in die cell extract as a 70 KDa protein. B) Expression in CHO cells (stable transformants) 1) The fusion protein lacking the membrane anchor domain. Fs±a The pEE14 -Fa" plasmid was transfected by calcium phosphate copredpitation into the CHO-K1 cells, using 20μg DNA per 1.25 10 6 cells cells. The CHO-Kl cells were grown in GMEM-S medium. The GS transfectants were selected by addition of 25μM metiiionine sulfoximine two days after transfection. After ten to fourteen days, resistant colonies were picked and transferred into 96 wells plates. Each transformant was then transferred into 24 wells plates and subsequendy to 80cm 2 flasks. The GS transformants were assayed for the Fs + a~ protein when cells reached about 80% confluency. The procedure follows the one described in Cockett, M.I., Bebbington, C.R. and Yarranton, G.T., Bio/Technology fi, 662-667, 1990. The Fs+a- protein was found in die spent culture medium of GS transformants. It is detected by Western blotting experiments using a rabbit polyclonal antibody raised against the purified mumps virion. Two bands are observed which constitute die two subunits of the protein (50 KDa and 20 KDa). 2) The hemagglutinin-neuraminidase lacking the 5' membrane anchor domain fused to the signal domain of the fusion protein. s+FHNa-. Procedures for expression of die s + FHNa" in CHO cells followed those described above. The s+FHNa- protein has been detected in d e spent culture medium by immunoprecipitation using a rabbit polyclonal antibody raised against die purified mumps virion; it appeared as a 84 KDa monomer. 3) The hybrid protein s+FHNa-xFa- Procedures for expression of die s+FHNa-xFa- in CHO cells followed tiiose described above. 4) The fusion protein lacking the 3' membrane anchor domain fused to the attachment protein lacking the 5' membrane anchor domain. Fs+a-xHNa- Procedures for expression of the Fs+a-xHNa- in CHO cells followed tiiose described above. C) Expression in Escherichia coli bacteria The NP protein Plasmid pNIV3217 was used to transform E.coli strain AR58 (MottJ.E., Grant,R., Ho.Y.Z. and Platt, T., 1985, Proc.Natl.Acad.Sci.USA £2, 88-92) which contains a defective lambda bacteriophage encoding die temperature-sensitive repressor, cI857ts. (pL transcription is repressed at low temperature but not at elevated temperature). Transformed bacteria were grown in 20ml of rich (LB) medium widi ampicillin (lOOμg/ml) up to an O.D. 630 of 0.6. Induction of die lambda P L promoter was achieved by shifting die temperature of the culture medium from 30°C to 42°C. One-milliliter aliquots of cultures were collected and centrifuged, pellets were resuspended in lOOμl 1% NaDodSO 4 , 6M urea, 5% 2-mercaptoethanol, and 10% glycerol, boiled for 5 min and lOμl samples were fractionated on 10% SDS- polyacrylamide gels. After transfer onto nitrocellulose membranes, separated proteins were probed widi a mixture of anti NP monoclonal antibodies (see supra Example II A.3 for references). A 69 KDa product corresponding to die NP protein was found exclusively in the induced sample. Example 3 Challenge experiments in the newborn hamster model The protective capacity of vaccinia recombinants, VV3205 (F protein), VV3208 (HN protein) and VV3213 (NP protein), was evaluated in the newborn hamster model as described previously (Overman ej a!-, 1953; Burr and Nagler, 1953; Love et al, 1985; Love el ai, 1986). The experiments proceeded in several steps: a) Female hamsters were infected by dermal scarification with each of die above vaccinia recombinants and widi a control vaccinia construct (2x10* pfii/animal). b) Antibody titers towards die specific proteins were measured periodically for several weeks after vaccination to ensure that an immune response was generated. c) Immune female hamsters were then mated to obtain newborn animals for the actual challenge experiment. d) Newborn hamsters were inoculated intracerebrally with the Kilham strain of Mumps virus (9.10 5 pfu per animal) and mortality due to encephalitis was followed for 10 days after challenge. Results Vaccinia recombinant HI Ab titer Overall survival in offspring after used for immunization in mothers challenge (10 days p.inf.) V V- β-lactamase <10 33.3% (5 surv./15 challenged) (control) VV3205 (F protein) 80 44.4% (4 surv. 9 challenged) VV3208 (HN protein) 80 100% (4 surv./4 challenged) VV3213 (NP protein) 10 2 15.8% (3 survJ19 challenged) VV3208 (HN) + 80 38.9% (7 survJlδ challenged) VV3205(F) The results show that VV3213 which expresses die NP protein does not reduce the mortality of newborn hamsters challenged with die Mumps virus. However, the mortality rate for newborn hamsters originating from hamster mothers immunized widi VV3205 (F) or 3208 (HN) was reduced, indicating tiiat the anti F and anti HN antibodies passed from the mothers to their offspring have the capacity to counteract the infection by the Mumps virus. Example 4: Purification of the HN protein of Mumps virus from recombinant vaccinia Virus NN 3208 Seventeen 150 cm2 flasks of CV1 cells were infected with W 3208 at a multiplicity of infection = 2. As a tracer for subsequent purification, one additional flask was supplemented widi 35S-medιionine. After 2 days of infection, cells were collected and lysed. Cell debris was removed by centrifugation and die clarified supernatant was passed on to an affinity column based on die monoclonal antibody Mab 2072. (10.8mg of antibody coupled to 1 ml of CΝBr - activated Sepaharose 4B, Pharmacia; protocols well known in the art). The recombinant HΝ protein binds to die affinity column and, after washing, is eluted from the matrix by 3M KSCΝ. The elution is monitored by counting aliquots labelled with incorporated 35S - methionine. Fractions containing the recombinant proteins were then pooled, dialysed to remove KSCΝ and lyophilised. The purity of the preparation was tested by migration on SDS - PAGE, gel-drying and autoradiography. The experiment shows that the recombinant HN protein displayed as expected a MW of about 80 KDa and that its purity exceeded 90%. The purification procedure yielded 250 ug of purified HN recombinant protein. This material can be used to boost the immune response in the animal model described above and, alternatively, to generate antibodies in mice and rabbits, providing useful tools for detection. References: Overman, J.R., Peers, J.H. and Kilham, L. (1953) Arch. Pathol. 55, 457-465. Burn, M.M. and Nagler, F.P. (1953) Proc.Soc.Exp.Biol.Med. £2, 714-717. Love, A., Rydbeck, R., Kristensson, K., Orvell,C. and Norrby,E. (1985) J.Virol. 58, 67-74. Love, A. i a! (1986) J.Virol. 59, 220-224.

Claims: Claims 1. Recombinant DNA encoding an antigen of mumps virus selected from the group comprising: (a) the full length fusion protein (F), complete with signal peptide (s) and membrane anchor domain (a) (designated herein as Fs+a+); die full length attachment protein or hemagglutinin-neuraminidase (HN); die full length nucleocapsid protein (NP); (b) the truncated fusion protein lacking the membrane anchor domain (herein designated Fs+a-); (c) a hybrid protein derived by fusion of die F, HN and/or NP proteins or portions thereof. 2. Recombinant DNA according to Claim 1 in which in groups (c) the hybrid protein is selected from: (i) die attachment protein lacking the 5' membrane anchor domain fused to die signal domain of die fusion protein (herein designated s+FHNa-); (ii) the hybrid protein herein designated s+FHNa-xFa-; (iii) the fusion protein lacking the 3' membrane anchor domain fused to die attachment protein lacking the 5' membrane anchor domain (herein designated Fs+a- xHNa-). 3. Recombinant DNA according to Claim 1 or Claim 2 which is a vector. 4. A vector according to Claim 3 which is derived from a vaccinia transfer vector. 5. A vector according to Claim 4 which is derived from pULB5212. 6. A vector according to Claim 5 which is selected from pNIV3205, pNTV3208, pNIV3213 and pNIV 3232. 7. A vector according to Claim 3 which is suitable for transfecting mammalian cells. 8. A vector according to Claim 7 which is derived from a glutamine synthetase 1o vector. 9. A vector according to Claim 8 which is derived from pEE14. 10. A vector according to Claim 3 which is suitable for transforming bacterial cells. 11. A vector according to Claim 10 which is derived from pUC19. 12. A host transformed or transfected wid a vector according to Claim 3. 13. A host according to Claim 12 selected from a vaccinia virus; a mammalian cell or a bacterial cell. 14. A method of preparing a recombinant protein encoded by DNA as claimed in Claim 1 or Claim 2 comprising: (a) expressing the said DNA in a recombinant host according to Claim 13; and (b) isolating die protein produced by standard techniques. 15. A protein selected from the group consisting of: a truncated fusion protein lacking the membrane anchor domain (herein designated Fs+a-); a hybrid protein derived by fusion of die F, HN and/or NP proteins or portions diereof. 16. A protein according to Claim 15 in which die hybrid protein is selected from: (i) the attachment protein lacking the 5' membrane anchor domain fused to die signal domain of die fusion protein (herein designated s+FHNa-); (ϋ) the hybrid protein herein designated s+FHNa-xFa-; (iii) the fusion protein lacking the 3' membrane anchor domain fused to the attachment protein lacking the 5' membrane anchor domain (herein designated Fs+a- xHNa-). 17. A vaccine composition comprising an immunoprotective amount of a protein prepared according to die method of Claim 14 or a protein according to Claim 15 or Claim 16 together with a pharmaceutically acceptable carrier. 18. A method of preventing mumps infections in humans which comprises administering to a human in need diereof an immunologically effective amount of a vaccine composition according to Claim 17.

Inventors: BOLLEN ALEX, HOUARD SOPHIE, NORRBY ERLING CARL JACOB, VARSANYI TAMAS MARK, BOLLEN, ALEX, HOUARD, SOPHIE, NORRBY, ERLING, CARL, JACOB, VARSANYI, TAMAS, MARK
Applicants: BOLLEN ALEX, HOUARD SOPHIE, NORRBY ERLING CARL JACOB, SMITHKLINE BEECHAM BIOLOG, VARSANYI TAMAS MARK, BOLLEN, ALEX, HOUARD, SOPHIE, NORRBY, ERLING, CARL, JACOB, SMITHKLINE BEECHAM BIOLOGICALS S.A., VARSANYI, TAMAS, MARK
Application year: 1994
Main classification: C12N15
IPC codes: A61K39, C07K14, C12N1, C12N15, C12R1